stripped memberane can be used for phosphprotein detection? - (Aug/23/2006 )

Hello,

I just wonder if the stripped blot can be used for phosphoprotein detection? The membrane was probed for twice already.

Thanks advance for any reply,

2manywestern

Hi,

I usually don't probe for phospho proteins if the blot has been stripped at all.

Typically, I probe for total protein first, strip, then probe for phospho protein. It sounds backwards, but yields the best results. Sometimes you will get a "masking effect" if you probe phospho first. Basically, this makes it appear as though you haven't loaded the same amount of protein per lane and is caused by differences in phospho vs non-phospho antibody affinities. Of course, this has been my experience with only a handful of proteins. The best way to answer your question is to test it out for yourself.

Hope that helps!
Hank

Usually I probe first with anti-phospho, strip, check the stripping by probing with the secondary antibody (ommiting the primary antibody), and then probe for total proteins, because my antibodies for total proteins give a better signal in any way than the antibodies directed against the phosphoproteins.
But you can also strip and probe with anti-phospho, it works if your signal is not too weak.

I do it the same way like missele, first anti-pospho, then total protein, then GAPDH. My phospho-Proteins give relatively weak signals, so better first. But depends on the kind of protein / antibody and your method of stripping.

Thanks a lot.

here a footnote: if one Ab is rabbit-polyclonal and the other Ab mouse-monoclonal, you may not strip if deactivating second Ab-peroxidase with NaN3 of the first IB round, then applying the second IB; better first monoclonal then polyclonal in this protocol

I've had phospho-antibodies that have worked after 2 or 3 strippings, it depends on expression levels, quality of antibody and the stripping buffer you use. The one I used that got me these results was 4M guanidine thiocyanate